Microbial Source Tracking and Fecal Indicator Bacteria TestingPathogen: will make you sickIndicator Bacteria: May or may not make you sickImportance of Clean BeachesEconomic:$2.5 bil CA Beach revenue$3.5 bil CA Ocean revenueEcosystem HealthFisheries, shellfish, recreation, etcHuman health1 billion beach trips annually, and >1 million affected nationally  $300 million health costsLA River: All storm drains, wastewater, etc runs down hereCA = strictest state for water testingAvalon has the worst oceanHouses have fresh and salt water come through the houseMost fresh water comes from a desalination plantSalt water is incredibly corrosiveSewer pipelines in Avalon were only red clay: there is no more sewage pipes !!Sewage constantly seeps out of pipesMalibu consistently gets bad gradesMany houses have septic tanks vs pipelines fecal indicator bacteria seeps into oceanMalibu lagoonDoes not flush  stale buildup of bird feces, shallow and warm  huge bloomsSit-ins because they think they will disrupt the organisms in the areaF beaches surrounded by A beachesOne area may b highly susceptibleBacteria may only be active in one areaSources of Fecal ContaminationLocal WildlifeSewage SpillsSeptic Tanks/LagoonsStorm DrainsHuntington: used bleach to clean up a huge sewage spill in 200810% of human body weight is bacteria, most of it is beneficialWater quality standards by EPA“Quality Criteria for Water”1976 Red book – first thoughts on water quality1986 Gold Book – Fecal indicator bacteria introduced; major breakthrough because related water quality and human healthClean Water Act (Federal Water Pollution Control Act)1972Multiple pollution control policiesStarts with the Rivers and Harbors Act of 1899Fecal Indicator BacteriaProkaryotesViruses predate on bacteriaIndicators of possible sewage contamination, commonly in human/animal fecesFecal coliforms (E.coli): specific to fecal matter from human/warm-blooded animalsEnterococci: resemble human pathogens, survive in saltwater; comes from humansTotal coliforms: widespread in nature; indicate contamination by outside source; not usually coming from humansHistory1986 Gold BookFIB counted vs sick peopleLinear relationshipTotal Maximum Daily Load Standards: State requirements can be more strict than the EPAHow often samples must be taken and what the TMDL isRegular monitoring must occur AND public must be informedHow much FIB is bad?Entero  1 org/mLFecal Col  4 org/mLTotal Col  10 org/mLCalifornia is leader in environmental regulationsTried to redraft the EPA standards, but people knew it was less safe and did not agree with itChange average from 30 days  90 days averageDidn’t changeApply standards to fresh and marine watersSample frequency responsibility of stateStates may require “rapid” methodsProblems with using fecal indicators to assess riskProduce false negatives/positives (Say it’s a problem but its not; vice-versa)E. Coli and Entero not only found in human wasteTime variation between when the water is sampled and when bacteria shows up on the petri dishCase Study at Two HarborsCat Harbor: long and narrow, not as much exchangeTerrible indicators in water and sandMore lax on lawsIsthmus Harbor: more open, able to have exchange with open oceanNo indicators in water, some on sandRapid methodsLess than one hour, for only the bacteria we are concerned aboutProbably wont happenPolymerase Chain Reaction (PCR)Highly sensitiveLooks for 1/16 of DNA that replicates that chunkTakes double stranded DNADNA Polymerase unzips the genes (Unzips the DNA)Add enzymes that are looking for the 16s sequence, then binds to it, and replicatesCools down and repeats, which amplifies the genesMust test for several fecal coliform bacteriaQuantitative PCRFluorescent marker comes up every time the 1/16 replicatesExtraction and amplification of target DNA  Use of fluorescent probes to visualize and quantify the environmental sampleTakes about half a day vs 1 hrMore replications  more fluorescent markers  easier to seeMicrobiology – cont. 02/07/2013Microbial Source Tracking and Fecal Indicator Bacteria Testing-Pathogen: will make you sick-Indicator Bacteria: May or may not make you sick Importance of Clean Beaches-Economic:o$2.5 bil CA Beach revenueo$3.5 bil CA Ocean revenue-Ecosystem HealthoFisheries, shellfish, recreation, etc-Human healtho1 billion beach trips annually, and >1 million affected nationally  $300 million health costs LA River: All storm drains, wastewater, etc runs down here CA = strictest state for water testing-Avalon has the worst ocean -Houses have fresh and salt water come through the house oMost fresh water comes from a desalination plantoSalt water is incredibly corrosiveSewer pipelines in Avalon were only red clay: there is nomore sewage pipes !! Sewage constantly seeps out of pipes -Malibu consistently gets bad grades oMany houses have septic tanks vs pipelines  fecal indicator bacteria seeps into oceanoMalibu lagoonDoes not flush  stale buildup of bird feces, shallow andwarm  huge bloomsSit-ins because they think they will disrupt the organisms in the area-F beaches surrounded by A beachesoOne area may b highly susceptible oBacteria may only be active in one areaSources of Fecal Contamination-Local Wildlife-Sewage Spills-Septic Tanks/Lagoons -Storm Drains-Huntington: used bleach to clean up a huge sewage spill in 200810% of human body weight is bacteria, most of it is beneficialWater quality standards by EPA- “Quality Criteria for Water”o1976 Red book – first thoughts on water qualityo1986 Gold Book – Fecal indicator bacteria introduced; major breakthrough because related water quality and human health-Clean Water Act (Federal Water Pollution Control Act)o1972oMultiple pollution control policiesoStarts with the Rivers and Harbors Act of 1899Fecal Indicator Bacteria-Prokaryotes-Viruses predate on bacteria-Indicators of possible sewage contamination, commonly in human/animal feces-Fecal coliforms (E.coli): specific to fecal matter from human/warm-blooded animals-Enterococci: resemble human pathogens, survive in saltwater; comes from humans -Total coliforms: widespread in nature; indicate contamination by outside source; not usually coming from humansHistory-1986 Gold Book-FIB counted vs sick people-Linear relationship-Total Maximum Daily Load Standards: State requirements can be more